The neural crest is a multipotent population of cells that has the ability to migrate throughout the embryo and give rise to a broad range of derivatives. Because of its contribution to multiple lineages, abnormal development of the NC can result in a wide array of seemingly unrelated clinical manifestations affecting multiple organ systems, as observed in Hirschsprung disease (hypopigmentation and aganglionic megacolon) and DiGeorge syndrome (craniofacial and heart defects). Therefore, studies focusing on the molecular mechanisms regulating the emergence of the NC are critical for furthering our understanding of a broad range of human congenital malformations and are the starting point for the development of new therapeutics that might serve to reverse these defects. In response to signaling events mediated by molecules of the Bmp, Wnt and Fgf families a number of transcription factors are sequentially induced at the neural plate border. First, a group of genes is activated, referred as neural plate border specifiers, which include members of the Zic, Pax, Dlx and Msx families of transcriptional regulators. These factors, which are broadly expressed at the neural plate border, are in turn responsible for the activation of a subset of genes with more restricted expression domains, known as NC specifiers among which are the SoxE proteins (Sox8, 9 and 10). Here we propose to analyze NC specification at three different levels in the regulatory cascade. 1- In Xenopus NC induction depends on a Bmp signal, which must be partially attenuated by Bmp antagonists, and a separate signal mediated by either a canonical Wnt or Fgf. While Bmp attenuation in the ectoderm appears to be a pre-requisite for NC induction, it is still unclear how Wnt and Fgf interact at the neural plate border to generate the NC. We propose to address the outstanding question of the relative contribution of Fgf and Wnt signaling to NC induction. 2- Morpholino-mediated knockdown of the neural plate border specifiers Pax3 or Zic1 indicates that these factors are both independently required for NC formation. Moreover, they synergistically activate NC fate. Our preliminary results indicate that by manipulating the levels of Pax3 and Zic1 in animal explants, we can generate NC progenitors independently of the induction of other neural plate border cell types. We propose to use the Pax3/Zic1 injected animal explants preparation to identify genes synergistically activated by Pax3/Zic1 in the developing NC. 3- Because ectopic expression of individual SoxE family members independently induces NC progenitors, it has been proposed that these factors are functionally equivalent. However, there is also evidence that SoxE proteins differentially regulate NC lineages. For example Sox9- and Sox10-deficient mice show severe but distinct NC defects suggesting that individual SoxE proteins play unique roles in NC development... We will define the common and unique functions of individual SoxE proteins during NC diversification by systematically analyzing the potential for individual SoxE proteins to rescue the NC phenotype of Sox8-, Sox9- or Sox10-depleted embryos. PUBLIC HEALTH RELEVANCE: Neural crest cells have the remarkable ability to contribute to a broad range of tissues in the embryo. Defects in the specification or differentiation of these cells may have very dramatic consequences on the development and function of many organ systems. Defining the factors that regulate the fate of these cells is critical to understand the molecular basis underlying these pathologies.